Nucleic acid amplification techniques and assays are well known. Some amplification reactions are isothermal, such as nucleic acid sequence based amplification (NASBA). Others employ thermal cycling, such as the polymerase chain reaction (PCR). Preferred amplification assays employing fluorescence detection of amplified product are homogeneous, that is, they do not require the physical separation of reagents to permit detection (for example, separation of bound probes from unbound probes) and can be performed in a single closed vessel. Such assays may be end-point, wherein product is detected after amplification, or they may be real-time, wherein product is detected as amplification proceeds.
Nucleic acid amplification and assays employing PCR are described, for example, in U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,965,188, and, generally, PCR PROTOCOLS, a guide to Methods and Applications, Innis et al. eds., Academic Press (San Diego, Calif. (USA) 1990). Homogeneous PCR assays, including real-time assays, in which amplified product is detected during some or all of the PCR cycles as the reaction proceeds are described, for example, in U.S. Pat. Nos. 5,994,056, 5,487,972, 5,925,517 and 6,150,097.
PCR amplification reactions generally are designed to be symmetric, that is, to make double-stranded amplicons exponentially by utilizing forward primer and reverse primer in equimolar concentrations and equal melting temperatures (Tm's). A technique that has found limited use for making single-stranded DNA directly in a PCR reaction is “asymmetric PCR.” Gyllensten and Erlich, “Generation of Single-Stranded DNA by the Polymerase Chain Reaction and Its Application to Direct Sequencing of the HLA-DQA Locus,” Proc. Natl. Acad. Sci. (USA) 85: 7652-7656 (1988); and U.S. Pat. No. 5,066,584. Asymmetric PCR is a non-symmetric PCR amplification method that differs from symmetric PCR in that one of the primers is diluted fivefold to one hundredfold so as to be present in limiting amount of 1-20 percent of the concentration of the other primer. As a consequence, the amplification consists of an exponential phase in which both primers are extended, generating double-stranded product, or amplicon, followed by a linear amplification in which only one primer remains, generating single-stranded amplicon.
More recently we have developed a non-symmetric PCR amplification method known as “Linear-After-The-Exponential” PCR or, for short, “LATE-PCR.” LATE-PCR is a non-symmetric PCR amplification consisting of an exponential phase in which both primers are annealed and extended followed by a linear phase after exhaustion of the Limiting Primer, when only the Excess Primer is annealed and extended. See Sanchez et al. (2004) Proc. Natl. Acad. Sci. (USA) 101: 1933-1938, published international patent application WO 03/054233 (3 Jul. 2003), and Pierce et al. (2005) Proc. Natl. Acad. Sci (USA) 102: 8609-8614, all of which are incorporated herein by reference in their entirety.
A convenient and inexpensive method for monitoring double-stranded amplicon production in a PCR amplification is to use a dye that fluoresces upon intercalating into or otherwise interacting with double-stranded DNA, such as SYBR Green I or SYBR Gold. See, for example, U.S. Pat. No. 5,994,056. Melting temperature analysis of amplicons performed either in real time during a PCR amplification or performed after amplification is used for product identification. One problem with utilizing such melting temperature analysis is that dye fluorescence is a function of amplicon size. Another problem is that dyes redistribute from amplification products, or amplicons, having low melting temperatures to amplicons having higher melting temperatures during analysis, thereby distorting results. Two approaches to solve these problems have been advanced. One approach, G quenching, imposes severe restrictions on primer design and causes large background fluorescence (Crockett A O, Wittwer C T. “Fluorescein-Labeled Oligonucleotides for Real-Time PCR: Using the Inherent Quenching of Deoxyguanosine Nucleotides” Anal. Biochem. 290:89-97 (2001)). The other, replacing SYBR dyes with LC Green dye, yields very small percentage of signal for sequences not present in abundance and requires highly specialized software and hardware (Wittwer et al. High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen,” Clin. Chem. 49:853-860 (2003).
Fluorescent-labeled probes are used in homogeneous nucleic acid amplification assays, including PCR assays, to measure the accumulation of desired amplicon, either in real time or by end-point analysis. Several available types of probes are significantly allele-discriminating as compared to linear single-stranded probes. Real-time probes include dual-labeled linear probes that are cleaved by 5′-to-3′ exonuclease activity of DNA polymerase during the extension step of a PCR cycle (see U.S. Pat. Nos. 5,210,015, 5,487,972 and 5,538,848); molecular beacon probes (see U.S. Pat. Nos. 5,925,517, 6,103,476 and 6,365,729); minor groove binding probes (see Afonina et al. “Minor Groove Binder-Conjugated DNA Probes for Quantitative DNA Detection by Hybridization-Triggered Fluorescence,” Biotechniques 32: 946-949 (2002)); linear probe pairs that FRET when hybridized adjacently on a target strand; quenched double-stranded linear probes for which a target competes to hybridize to the labeled probe strand (see Li, Q. et al. (2002), Nucl. Acid. Res. 30: e5); and so-called “light-up” probes, which are peptide nucleic acid (PNA) oligomers linked to an asymmetric cyanine dye that fluoresces when the probe binds to target to form a double-stranded region. For LATE-PCR we have utilized low-temperature allele-discriminating probes, such as low temperature molecular beacon probes (See WO 03/045233). Labeled oligonucleotide probes may be attached to primers by linkers such that during amplification the probes are not copied but are free to hybridize to a target sequence resulting from extension of the primer. Examples are Scorpions®, primers to which are attached molecular beacon probes, and Anglers®, primers to which are attached fluorophore-labeled linear probes. Lee, M. A. et al. (2002), Analytica Clinica Acta 457: 61:70; Whitcombe, D. et al. (1999), Nature Biotechnology 17: 804-807. The probe portion of such composite structures, which carries the fluorescent label, hybridizes separately from the primer portion. They are, thus, labeled probes and not labeled primers, as those terms are used herein. Target-specific probes lack the capacity to monitor total production of double-stranded products, however.
Certain probes are mismatch-tolerant. Mismatch-tolerant probes hybridize with and generate detectable signal for more than one target sequence at a detection temperature in an assay, and various hybrids so formed will have different melting points. Linear, or random coil, single-stranded probes are generally mismatch tolerant. Examples of such probes are linear probes with an internal fluorescent moiety whose level of fluorescence increases upon hybridization to one or another target strand; fluorescently labeled linear probes used in combination with SYBR Gold and SYBR Green I dyes, such that fluorescence of the label occurs by FRET from the dye when the probe hybridizes to one or another target (see U.S. patent publication US 2002/0119450, 28 Aug. 2002), so-called “sloppy beacons”, and variations of linear probe pairs that FRET (see U.S. Pat. No. 6,472,156).
Utilizing multiple probes that each bind only to one possible target amplicon generated in an amplification reaction creates a problem for analyzing complicated reaction mixtures or for detecting one or a few targets from among numerous possible targets. Available fluorescence detection permits resolution of a limited number of fluorophores, generally no more than eight. Limited multiplexing is possible, for example, by designing a different allele-discriminating molecular beacon probe for each target and labeling each probe differentially. (See, for example, Tyagi et al. (2000) Nature Biotechnology 18: 1191-1196). Mixtures of allele-discriminating probes, each comprising aliquots of multiple colors, extends the number of probe signatures and works well if only one of many (at least up to 56) targets is actually present, but it encounters ambiguous results if more than one target is present.
There are many situations that involve complex targets or one among many possible targets. Several schemes have been developed or proposed for such situations, but all have serious drawbacks and limitations. Tyagi et al. published international patent application WO 01/31062, have described a technique sometimes referred to as “sloppy beacons,” which are molecular beacon probes that have long loop sequences, rendering them mismatch tolerant and able to bind to some extent to multiple targets at the annealing temperature of a PCR amplification reaction. Such probes suffer from poor reaction kinetics against mismatched targets and are likely to remain hybridized to perfectly matched targets at the extension temperature of a PCR amplification and be cleaved by Taq DNA polymerase. Further, only an indirect indication of melting temperatures of probe-target hybrids under the assay conditions is obtained, and that assumes equilibrium has been achieved. Real-time multiplexing in symmetric PCR amplifications with FRET probes has been described. In order not to interfere with amplification, the melting temperatures of all probe-target hybrids are constrained to be in the narrow temperature range between the primer annealing temperature and the primer extension temperature. Also, that scheme is not quantitative. Post-amplification multiprobe assays employing FRET probes of different colors have been disclosed Wittwer et al., “Real-Time Multiplex PCR Assays, Methods” 25:430-442 (2001). The reaction mixture following a symmetric PCR amplification is rapidly chilled, then slowly heated to determine melting curves for the various fluorophores present. This approach is not quantitative. In addition, because of large scatter among replicate symmetric PCR amplifications, end-point assays in general tend to be only qualitative.
Sequencing reaction products provides an alternative to probing. Traditional dideoxy sequencing may utilize products of amplification reactions, such as symmetric PCR or LATE-PCR, as starting materials for cycle sequencing. Amplified product is purified utilizing ethanol precipitation or an affinity column to remove leftover dNTPs and primers, subjected to a cycle sequencing reaction utilizing one sequencing primer and fluorescently labeled dideoxy nucleotides, and subjected to capillary gel electrophoresis. “Pyrosequencing” is a real-time, isothermal, sequence-by-synthesis method known in the art. If exponential amplification methods, for example PCR, are used in the preparation of starting material for Pyrosequencing, amplified product must be cleaned up by isolation of single-stranded product as well as removal of dNTPs, pyrophosphate and unincorporated primers from the amplification reaction. LATE-PCR simplifies sample preparation, because it generates primarily single-stranded product, but it does not in and of itself eliminate the need to clean-up the product.
An aspect of this invention is methods for homogeneous detection of reaction products of amplification reactions, temperature cycling or isothermal, utilizing the detection of fluorescence from fluorophore-labeled linear oligonucleotide primers excited indirectly by exciting a DNA fluorescent dye such as SYBR Green I or, preferably, SYBR Gold. Such dyes become fluorescent when they associate with double-stranded DNA, into which they are reported to intercalate. The foregoing methods may be performed in real time or following the amplification reaction, either by reading fluorescence at a detection temperature (end-point detection) or by ascertaining changes in fluorescence as a function of temperature by post-amplification melting analysis. As a reaction mixture is heated through the melting temperatures of various reaction products, fluorescence decreases progressively as various amplicons containing a particular fluorophore-containing primer reach their melting temperatures and become single-stranded. Preferred methods include calculating the ratio of primer signal to dye signal.
Another aspect of this invention is reagent kits that include both DNA fluorescents dye and at least one such labeled primer, preferably as part of a primer pair, and optionally amplification reagents.
Yet other aspects of this invention are homogeneous methods for detecting reaction products of LATE-PCR reactions employing a low-temperature detection step. Certain embodiments comprise including in the reaction mixture at least one allele-discriminating probe according to this invention, namely, a quenched double-stranded probe generally of the type described by Li, Q. et al. (2002) Nucl. Acids Res. 30: e5 except that it is a low temperature (Low-Tm, or Super-Low Tm) target-specific probe and that it is excited indirectly by exciting a DNA fluorescent dye intercalated into the probe-target hybrid such as, preferably, SYBR Gold. Other embodiments comprise including in the reaction mixture at least one indirectly excitable mismatch-tolerant probe according to this invention, namely, a quenched single-stranded probe generally of the type described by Lee and Furst United States published patent application Pub. No. US 2002/0119450 except that is a quenched low-temperature probe. These various methods include exciting the dye during the low-temperature detection steps of a LATE-PCR amplification and detecting fluorescence from the probes under these conditions to provide a measure of the target single-stranded sequence. Particular embodiments may further include measuring the total amount of double-stranded product(s) in the reaction mixture by detecting dye fluorescence, preferably during or at the end of the extension step of PCR cycles while the temperature of the reaction mixture is above the melting temperature(s) of the probes. Certain preferred methods include calculating the ratio of probe signal to dye signal. In the case of replicate samples, such ratio corrects for differences among replicate samples in amplification yields known to occur in PCR amplifications.
Other aspects of this invention are such low-temperature allele-discriminating and quenched mismatch-tolerant probes, LATE-PCR kits that include at least one such low-temperature target-specific probe together with amplification reagents and preferably the fluorescent DNA dye; and oligonucleotide sets comprising LATE-PCR primers and at least one such probe.
Other aspects of this invention are homogeneous detection methods for use when multiple amplicons are present or may be present, such method comprising including in a LATE-PCR amplification reaction mixture one or more low-temperature mismatch-tolerant detection probes that, because of their low Tm, do not interfere with amplification and are not hydrolysed (cut) by a DNA polymerase having 5′-3′ exonuclease activity, and that emit a fluorescent signal when hybridized and excited, either directly by a suitable excitation source or indirectly by a fluorescent DNA dye that is excited by a suitable excitation source. Such methods include single-probe assays and multiple-probe assays for applications such as genotyping. More than one probe may be labeled with the same fluorophore, in which event discrimination relies on change in fluorescence with temperature, just as when a single probe is used. Probes may be labeled with different fluorophores, in which event color difference is also used for discrimination. Discrimination among targets for purposes of identification and quantification may include fluorescence ratios between fluorophores at the same or different temperatures, as well as fluorophore-to-dye ratios. Detection is preferably performed during the amplification (real time) and more preferably during a low-temperature detection step included in a LATE-PCR amplification protocol, and the detection step may include detection at multiple temperatures. Yet another aspect of this invention is a single-stranded linear probe useful in such detection methods, such probe being of the type described in U.S. patent application publication U.S. 2002/0119450 (29 Aug. 2002), that is, a probe excited by the fluorescence emission from a fluorescent DNA dye, except that it is a low-temperature (Low-Tm or Super-Low-Tm) probe, is mismatch-tolerant, and includes a quenching moiety that quenches the fluorescence, which otherwise would result from secondary structure of at low temperature.
Another aspect of this invention is an amplification-through-sequencing method that permits the product of a LATE-PCR amplification to be prepared for pyrosequencing in the amplification reaction chamber, vessel, well, slide or container, a “single-tube” operation, which may be utilized with LATE-PCR amplifications performed in small volumes, preferably 17 ul or less.
Another aspect of this invention is a method for preparing LATE-PCR products for dideoxy sequencing utilizing only post-amplification aqueous dilution of amplification reaction mixtures, which may be performed as a “single-tube” operation.